TM4SF5 Antibody

What is TM4SF5 and in which cancers is it primarily expressed?

TM4SF5 (transmembrane four L6 family member 5) is a tetraspanin-like protein that is aberrantly expressed in multiple cancer types. Based on immunohistochemical analyses, TM4SF5 is highly expressed in liver (hepatocellular carcinoma), colon, esophageal, and pancreatic cancers . Expression analysis of colon cancer tissues revealed that nearly all samples (44 out of 45, or 97.8%) expressed TM4SF5, with approximately 35.6% of samples showing TM4SF5 expression in ≥75% of tumor cells and 40% showing expression in 50-74% of tumor cells . In contrast, normal colon tissues showed no TM4SF5 expression, suggesting its potential as a cancer-specific marker .

How does TM4SF5 contribute to tumor progression at the molecular level?

TM4SF5 contributes to tumor progression through multiple molecular mechanisms. It disrupts cell-cell adhesion by inducing the epithelial-mesenchymal transition (EMT), characterized by decreased E-cadherin and increased expression of mesenchymal markers . TM4SF5 causes mislocalization of p27kip1 from the nucleus to the cytosol and reduces RhoA activity, leading to uncontrolled cell growth and tumorigenesis through loss of contact inhibition . Additionally, TM4SF5 accelerates G1/S phase transition by controlling cytosolic p27kip1 and RhoA activity, further promoting cancer cell proliferation . TM4SF5 also induces vascular endothelial growth factor (VEGF) expression and secretion, enhancing angiogenic activity that supports tumor growth .

What approaches have been used to develop effective anti-TM4SF5 monoclonal antibodies?

Researchers have successfully developed anti-TM4SF5 monoclonal antibodies using phage display technology. Specifically, a mouse single-chain variable fragment (scFv) library was screened to identify antibodies that specifically bind to the extracellular loop 2 (EC2) domain of human TM4SF5 . The identified antibodies were then used to generate scFv-human Fc chimeric antibodies, designated Ab27 and Ab79 . For humanization, the complementarity-determining region (CDR) grafting method was employed to reduce immunogenicity while maintaining target recognition and antitumor activity. This process resulted in the humanized antibody Ab27-hz9, which exhibited comparable binding specificity and anti-cancer activity to the original Ab27 .

What methodologies are recommended for validating the specificity of anti-TM4SF5 antibodies?

Several methodologies are essential for validating anti-TM4SF5 antibody specificity:

• Flow cytometry: This technique can confirm antibody binding to TM4SF5-expressing cells. Researchers have validated antibody specificity by comparing binding to TM4SF5-expressing cells versus cells transfected with TM4SF5-specific siRNA. Typical protocols involve incubating cells (2 × 10^5) with antibodies at concentrations between 0.3-1 μg/ml, followed by detection with fluorescently-labeled secondary antibodies .

• Knockdown experiments: Comparing antibody binding in cells with and without TM4SF5 knockdown via siRNA provides strong evidence of specificity .

• Immunohistochemistry: This technique validates antibody specificity by comparing staining patterns in cancer tissues (which express TM4SF5) versus normal tissues (which typically do not) .

• Western blotting: Analyzing protein expression before and after antibody treatment can confirm target engagement and downstream effects on signaling pathways .

How can TM4SF5 expression be quantified in patient tumor samples?

TM4SF5 expression in patient tumor samples can be effectively quantified using immunohistochemistry with anti-TM4SF5 monoclonal antibodies. Researchers have developed a systematic scoring system based on the percentage of tumor cells expressing TM4SF5, categorized into four levels:

In a comprehensive analysis of colon cancer tissues (n=45), 35.6% showed high TM4SF5 expression, 40.0% showed moderate expression, 22.2% showed low expression, and only 2.2% were negative . This standardized approach facilitates consistent evaluation across different research settings and potential correlation with clinical outcomes.

What cellular assays are most informative for assessing the functional effects of anti-TM4SF5 antibodies?

Several cellular assays have proven particularly informative for assessing anti-TM4SF5 antibody effects:

• Cell viability/proliferation assays: These measure the direct anti-proliferative effects of antibodies on TM4SF5-expressing cancer cells, providing quantitative data on growth inhibition .

• Cell motility assays: Since TM4SF5 promotes cancer cell motility, wound healing and transwell migration assays can evaluate how anti-TM4SF5 antibodies affect this cancer-promoting property .

• EMT marker analysis: Measuring changes in EMT markers (E-cadherin, vimentin, β-catenin) after antibody treatment provides insights into the reversal of the mesenchymal phenotype. Anti-TM4SF5 antibody treatment has been shown to enhance expression of E-cadherin and β-catenin in colon cancer cells .

• Signaling pathway analysis: Western blotting to detect changes in phosphorylation of focal adhesion kinase (FAK), p27kip1, and signal transducer and activator of transcription 3 (STAT3) after antibody treatment reveals the molecular mechanisms of action .

Through what molecular mechanisms do anti-TM4SF5 antibodies exert their anti-cancer effects?

Anti-TM4SF5 antibodies exert their anti-cancer effects through multiple molecular mechanisms:

• Reversal of EMT: Anti-TM4SF5 antibodies modulate the expression of EMT markers, decreasing vimentin (mesenchymal marker) and increasing E-cadherin (epithelial marker), effectively reversing the mesenchymal phenotype that promotes cancer cell invasion and metastasis .

• Nuclear translocation of p27kip1: Treatment with anti-TM4SF5 antibodies induces translocation of p27kip1 from the cytosol back to the nucleus, restoring its cell cycle inhibitory function .

• RhoA activation: Anti-TM4SF5 antibodies increase RhoA activity, which helps restore normal cell-cell contacts and contact inhibition .

• Inhibition of key signaling pathways: Ab27 treatment decreases the phosphorylation of focal adhesion kinase (FAK), p27kip1, and signal transducer and activator of transcription 3 (STAT3), disrupting cancer-promoting signaling cascades .

• Immune cell-mediated killing: Beyond direct effects on cancer cells, antibodies like Ab27 exhibit immune cell-mediated killing activity, potentially engaging the host immune system against cancer cells .

How do the effects of anti-TM4SF5 antibodies differ between in vitro and in vivo cancer models?

The effects of anti-TM4SF5 antibodies show important distinctions between in vitro and in vivo cancer models:

In vitro effects:

• Direct inhibition of cancer cell proliferation

• Reduction in cell viability

• Modulation of EMT markers

• Decreased cell motility

• Molecular changes in signaling pathways

In vivo effects:

• Significant reduction of tumor growth in xenograft and allograft models

• Efficacy in multiple cancer types (liver, colon)

• Effectiveness against sorafenib-resistant tumors

• Enhanced anti-tumor activity when combined with conventional chemotherapeutics (sorafenib, doxorubicin)

• No general toxicity observed in animal models

• Efficient targeting of TM4SF5-expressing tumor cells

Importantly, the humanized antibody Ab27-hz9 maintained comparable in vivo anti-tumor activity to the chimeric Ab27 while demonstrating reduced immunogenicity, highlighting its potential for clinical translation .

What strategies have been developed for antibody humanization to improve clinical applicability?

The complementarity-determining region (CDR) grafting method has been successfully employed to humanize anti-TM4SF5 antibodies. Specifically, the development of Ab27-hz9 followed this approach:

• The variable heavy (VH) and variable kappa light (Vk) chain encoding genes from the original murine antibody were synthesized .

• These genes were inserted into modified expression vectors carrying the human IgG1 constant regions (CH1-hinge-CH2-CH3) or human kappa chain constant region (CL) .

• The constructs were expressed in HEK 293F cells, followed by purification using Protein A affinity chromatography .

This humanization process resulted in Ab27-hz9, which showed reduced immunogenicity while maintaining target recognition and anti-tumor activity comparable to the original Ab27 . The humanized antibody efficiently targeted TM4SF5-expressing tumor cells in vivo, supporting its development as a therapeutic agent .

What combination therapies with anti-TM4SF5 antibodies have shown enhanced anti-cancer efficacy?

Combination therapies with anti-TM4SF5 antibodies have demonstrated superior anti-cancer efficacy compared to monotherapies:

• Ab27 + Sorafenib: This combination exerted higher antitumor activity than either agent alone, suggesting a potential strategy to enhance the efficacy of sorafenib, a multikinase inhibitor approved for hepatocellular carcinoma .

• Ab27 + Doxorubicin: Combining Ab27 with the chemotherapeutic agent doxorubicin also showed enhanced anti-tumor effects compared to monotherapy .

These findings are particularly significant because:

• The combinations were effective even against sorafenib-resistant tumor models, suggesting a potential approach to overcome treatment resistance .

• No general toxicity was observed with Ab27 in vivo, indicating a favorable safety profile for combination approaches .

The synergistic effects observed with these combinations suggest that anti-TM4SF5 antibodies may sensitize cancer cells to conventional therapeutics by modulating TM4SF5-mediated signaling pathways.

What are the emerging research areas for TM4SF5 antibodies beyond hepatocellular and colorectal cancers?

While much of the current research has focused on hepatocellular and colorectal cancers, several emerging research areas for anti-TM4SF5 antibodies warrant further investigation:

• Pancreatic cancer: Studies have demonstrated that TM4SF5 is expressed in pancreatic cancer cells and that anti-TM4SF5 antibody treatment reduces cell viability and motility in these cells . The high mortality rate of pancreatic cancer and limited treatment options make this an especially promising area for continued research.

• Esophageal cancer: TM4SF5 is highly expressed in esophageal cancer , but the therapeutic potential of anti-TM4SF5 antibodies in this cancer type remains incompletely explored.

• Combination with immunotherapy: Given that anti-TM4SF5 antibodies like Ab27 exhibit immune cell-mediated killing activity , investigating combinations with immune checkpoint inhibitors could potentially enhance therapeutic efficacy.

• Development of antibody-drug conjugates: Conjugating cytotoxic payloads to anti-TM4SF5 antibodies could potentially enhance their direct tumor-killing capacity while maintaining their targeting specificity.

What methodological considerations are important when evaluating anti-TM4SF5 antibodies in treatment-resistant cancer models?

When evaluating anti-TM4SF5 antibodies in treatment-resistant cancer models, several methodological considerations are crucial:

• Appropriate resistance model selection: Researchers have successfully used sorafenib-resistant models to demonstrate the efficacy of Ab27 . Selection of clinically relevant resistance models is essential for translational impact.

• Combination treatment design: When testing anti-TM4SF5 antibodies in combination with standard therapies, careful consideration of dosing schedules (sequential vs. concurrent) and dose ratios is necessary to optimize synergistic effects.

• Mechanistic validation: Understanding how TM4SF5 expression and signaling may differ in resistant versus sensitive cells provides insights into why anti-TM4SF5 antibodies remain effective when other therapies fail.

• Biomarker identification: Identifying predictive biomarkers that correlate with response to anti-TM4SF5 antibody therapy in resistant models would facilitate patient selection in clinical applications.

• Long-term treatment effects: Assessing whether resistance to anti-TM4SF5 antibodies develops over time, and the mechanisms underlying such resistance, is critical for anticipating clinical challenges.

The promising results with Ab27 in sorafenib-resistant models suggest that targeting TM4SF5 may represent an effective strategy for overcoming treatment resistance in multiple cancer types.